Mammalian Genome

, Volume 24, Issue 5–6, pp 206–217 | Cite as

No evidence for cumulative effects in a Dnmt3b hypomorph across multiple generations

  • Neil A. Youngson
  • Trevor Epp
  • Amity R. Roberts
  • Lucia Daxinger
  • Alyson Ashe
  • Edward Huang
  • Krystal L. Lester
  • Sarah K. Harten
  • Graham F. Kay
  • Timothy Cox
  • Jacqueline M. Matthews
  • Suyinn Chong
  • Emma WhitelawEmail author


Observations of inherited phenotypes that cannot be explained solely through genetic inheritance are increasing. Evidence points to transmission of non-DNA molecules in the gamete as mediators of the phenotypes. However, in most cases it is unclear what the molecules are, with DNA methylation, chromatin proteins, and small RNAs being the most prominent candidates. From a screen to generate novel mouse mutants of genes involved in epigenetic reprogramming, we produced a DNA methyltransferase 3b allele that is missing exon 13. Mice that are homozygous for the mutant allele have smaller stature and reduced viability, with particularly high levels of female post-natal death. Reduced DNA methylation was also detected at telocentric repeats and the X-linked Hprt gene. However, none of the abnormal phenotypes or DNA methylation changes worsened with multiple generations of homozygous mutant inbreeding. This suggests that in our model the abnormalities are reset each generation and the processes of transgenerational epigenetic reprogramming are effective in preventing their inheritance.


Green Fluorescent Protein Expression Bisulphite Sequencing Transgenerational Epigenetic Inheritance Genomic Imprint Litter Size Reduction 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The authors thank En Li (China Novartis Institutes for BioMedical Research) for the Dnmt3b knockout mice. This study was supported by Australian NHMRC project grants to EW. ARR and KLL were supported by Australian Postgraduate awards. JMM is supported by an NHMRC Senior Research Fellowship. EW is supported by an NHMRC Australia Fellowship.

Supplementary material

335_2013_9451_MOESM1_ESM.doc (234 kb)
Supplementary material 1 (DOC 234 kb)


  1. Argentaro A, Yang JC, Chapman L, Kowalczyk MS, Gibbons RJ, Higgs DR, Neuhaus D, Rhodes D (2007) Structural consequences of disease-causing mutations in the ATRX-DNMT3-DNMT3L (ADD) domain of the chromatin-associated protein ATRX. Proc Natl Acad Sci USA 104:11939–11944PubMedCrossRefGoogle Scholar
  2. Ashe A, Morgan DK, Whitelaw NC, Bruxner TJ, Vickaryous NK, Cox LL, Butterfield NC, Wicking C, Blewitt ME, Wilkins SJ, Anderson GJ, Cox TC, Whitelaw E (2008) A genome-wide screen for modifiers of transgene variegation identifies genes with critical roles in development. Genome Biol 9:R182PubMedCrossRefGoogle Scholar
  3. Biniszkiewicz D, Gribnau J, Ramsahoye B, Gaudet F, Eggan K, Humpherys D, Mastrangelo MA, Jun Z, Walter J, Jaenisch R (2002) Dnmt1 overexpression causes genomic hypermethylation, loss of imprinting, and embryonic lethality. Mol Cell Biol 22:2124–2135PubMedCrossRefGoogle Scholar
  4. Blewitt ME, Vickaryous NK, Hemley SJ, Ashe A, Bruxner TJ, Preis JI, Arkell R, Whitelaw E (2005) An N-ethyl-N-nitrosourea screen for genes involved in variegation in the mouse. Proc Natl Acad Sci USA 102:7629–7634PubMedCrossRefGoogle Scholar
  5. Bock C, Reither S, Mikeska T, Paulsen M, Walter J, Lengauer T (2005) BiQ Analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing. Bioinformatics 21:4067–4068PubMedCrossRefGoogle Scholar
  6. Bohacek J, Gapp K, Saab BJ, Mansuy IM (2013) Transgenerational epigenetic effects on brain functions. Biol Psychiatry 73(4):313–320PubMedCrossRefGoogle Scholar
  7. Bourc’his D, Miniou P, Jeanpierre M, Molina Gomes D, Dupont J, De Saint-Basile G, Maraschio P, Tiepolo L, Viegas-Pequignot E (1999) Abnormal methylation does not prevent X inactivation in ICF patients. Cytogenet Cell Genet 84:245–252PubMedCrossRefGoogle Scholar
  8. Brykczynska U, Hisano M, Erkek S, Ramos L, Oakeley EJ, Roloff TC, Beisel C, Schubeler D, Stadler MB, Peters AH (2010) Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa. Nat Struct Mol Biol 17:679–687PubMedCrossRefGoogle Scholar
  9. Chong S, Vickaryous N, Ashe A, Zamudio N, Youngson N, Hemley S, Stopka T, Skoultchi A, Matthews J, Scott HS, de Kretser D, O’Bryan M, Blewitt M, Whitelaw E (2007) Modifiers of epigenetic reprogramming show paternal effects in the mouse. Nat Genet 39:614–622PubMedCrossRefGoogle Scholar
  10. Danchin E, Charmantier A, Champagne FA, Mesoudi A, Pujol B, Blanchet S (2011) Beyond DNA: integrating inclusive inheritance into an extended theory of evolution. Nat Rev Genet 12:475–486PubMedCrossRefGoogle Scholar
  11. Daxinger L, Whitelaw E (2012) Understanding transgenerational epigenetic inheritance via the gametes in mammals. Nat Rev Genet 13:153–162PubMedCrossRefGoogle Scholar
  12. Daxinger L, Oey H, Apedaile A, Sutton J, Ashe A, Whitelaw E (2012) A forward genetic screen identifies eukaryotic translation initiation factor 3, subunit H (eIF3h), as an enhancer of variegation in the mouse. G3 (Bethesda) 2:1393–1396CrossRefGoogle Scholar
  13. Dhayalan A, Tamas R, Bock I, Tattermusch A, Dimitrova E, Kudithipudi S, Ragozin S, Jeltsch A (2011) The ATRX-ADD domain binds to H3 tail peptides and reads the combined methylation state of K4 and K9. Hum Mol Genet 20:2195–2203PubMedCrossRefGoogle Scholar
  14. Gartler SM, Varadarajan KR, Luo P, Canfield TK, Traynor J, Francke U, Hansen RS (2004) Normal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins. BMC Biol 2:21PubMedCrossRefGoogle Scholar
  15. Gendrel AV, Apedaile A, Coker H, Termanis A, Zvetkova I, Godwin J, Tang YA, Huntley D, Montana G, Taylor S, Giannoulatou E, Heard E, Stancheva I, Brockdorff N (2012) Smchd1-dependent and -independent pathways determine developmental dynamics of CpG island methylation on the inactive X chromosome. Dev Cell 23:265–279PubMedCrossRefGoogle Scholar
  16. Grandjean V, Gounon P, Wagner N, Martin L, Wagner KD, Bernex F, Cuzin F, Rassoulzadegan M (2009) The miR-124-Sox9 paramutation: RNA-mediated epigenetic control of embryonic and adult growth. Development 136:3647–3655PubMedCrossRefGoogle Scholar
  17. Guenatri M, Bailly D, Maison C, Almouzni G (2004) Mouse centric and pericentric satellite repeats form distinct functional heterochromatin. J Cell Biol 166:493–505PubMedCrossRefGoogle Scholar
  18. Guerrero-Bosagna C, Skinner MK (2012) Environmentally induced epigenetic transgenerational inheritance of phenotype and disease. Mol Cell Endocrinol 354:3–8PubMedCrossRefGoogle Scholar
  19. Guerrero-Bosagna C, Settles M, Lucker B, Skinner MK (2010) Epigenetic transgenerational actions of vinclozolin on promoter regions of the sperm epigenome. PLoS One 5(9):e13100. doi: 10.1371/journal.pone.0013100
  20. Guibert S, Forne T, Weber M (2012) Global profiling of DNA methylation erasure in mouse primordial germ cells. Genome Res 22:633–641PubMedCrossRefGoogle Scholar
  21. Hadchouel M, Farza H, Simon D, Tiollais P, Pourcel C (1987) Maternal inhibition of hepatitis B surface antigen gene expression in transgenic mice correlates with de novo methylation. Nature 329:454–456PubMedCrossRefGoogle Scholar
  22. Hagleitner MM, Lankester A, Maraschio P, Hulten M, Fryns JP, Schuetz C, Gimelli G, Davies EG, Gennery A, Belohradsky BH, de Groot R, Gerritsen EJ, Mattina T, Howard PJ, Fasth A, Reisli I, Furthner D, Slatter MA, Cant AJ, Cazzola G, van Dijken PJ, van Deuren M, de Greef JC, van der Maarel SM, Weemaes CM (2008) Clinical spectrum of immunodeficiency, centromeric instability and facial dysmorphism (ICF syndrome). J Med Genet 45:93–99PubMedCrossRefGoogle Scholar
  23. Hammoud SS, Nix DA, Zhang H, Purwar J, Carrell DT, Cairns BR (2009) Distinctive chromatin in human sperm packages genes for embryo development. Nature 460:473–478Google Scholar
  24. Hansen RS, Wijmenga C, Luo P, Stanek AM, Canfield TK, Weemaes CM, Gartler SM (1999) The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome. Proc Natl Acad Sci USA 96:14412–14417PubMedCrossRefGoogle Scholar
  25. Heyn H, Vidal E, Sayols S, Sanchez-Mut JV, Moran S, Medina I, Sandoval J, Simo-Riudalbas L, Szczesna K, Huertas D, Gatto S, Matarazzo MR, Dopazo J, Esteller M (2012) Whole-genome bisulfite DNA sequencing of a DNMT3B mutant patient. Epigenetics 7:542–550PubMedCrossRefGoogle Scholar
  26. Jin B, Tao Q, Peng J, Soo HM, Wu W, Ying J, Fields CR, Delmas AL, Liu X, Qiu J, Robertson KD (2008) DNA methyltransferase 3B (DNMT3B) mutations in ICF syndrome lead to altered epigenetic modifications and aberrant expression of genes regulating development, neurogenesis and immune function. Hum Mol Genet 17:690–709PubMedCrossRefGoogle Scholar
  27. Juriloff DM, Harris MJ (2012) Hypothesis: the female excess in cranial neural tube defects reflects an epigenetic drag of the inactivating X chromosome on the molecular mechanisms of neural fold elevation. Birth Defects Res A Clin Mol Teratol 94:849–855PubMedCrossRefGoogle Scholar
  28. Kalitsis P, Griffiths B, Choo KH (2006) Mouse telocentric sequences reveal a high rate of homogenization and possible role in Robertsonian translocation. Proc Natl Acad Sci USA 103:8786–8791PubMedCrossRefGoogle Scholar
  29. Kaneda M, Okano M, Hata K, Sado T, Tsujimoto N, Li E, Sasaki H (2004) Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature 429:900–903PubMedCrossRefGoogle Scholar
  30. Li E, Bestor TH, Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69:915–926PubMedCrossRefGoogle Scholar
  31. Li JY, Lees-Murdock DJ, Xu GL, Walsh CP (2004) Timing of establishment of paternal methylation imprints in the mouse. Genomics 84:952–960PubMedCrossRefGoogle Scholar
  32. Liu WM, Pang RT, Chiu PC, Wong BP, Lao K, Lee KF, Yeung WS (2012) Sperm-borne microRNA-34c is required for the first cleavage division in mouse. Proc Natl Acad Sci USA 109:490–494PubMedCrossRefGoogle Scholar
  33. Morgan HD, Sutherland HG, Martin DI, Whitelaw E (1999) Epigenetic inheritance at the agouti locus in the mouse. Nat Genet 23:314–318PubMedCrossRefGoogle Scholar
  34. Nelson VR, Nadeau JH (2010) Transgenerational genetic effects. Epigenomics 2:797–806PubMedCrossRefGoogle Scholar
  35. Neuhaus IM, Beier DR (1998) Efficient localization of mutations by interval haplotype analysis. Mamm Genome 9(2):150–154PubMedCrossRefGoogle Scholar
  36. Okano M, Bell DW, Haber DA, Li E (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99:247–257PubMedCrossRefGoogle Scholar
  37. Ooi SK, Qiu C, Bernstein E, Li K, Jia D, Yang Z, Erdjument-Bromage H, Tempst P, Lin SP, Allis CD, Cheng X, Bestor TH (2007) DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA. Nature 448:714–717PubMedCrossRefGoogle Scholar
  38. Otani J, Nankumo T, Arita K, Inamoto S, Ariyoshi M, Shirakawa M (2009) Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX-DNMT3-DNMT3L domain. EMBO Rep 10:1235–1241PubMedCrossRefGoogle Scholar
  39. Preis JI, Downes M, Oates NA, Rasko JE, Whitelaw E (2003) Sensitive flow cytometric analysis reveals a novel type of parent-of-origin effect in the mouse genome. Curr Biol 13:955–959PubMedCrossRefGoogle Scholar
  40. Rassoulzadegan M, Grandjean V, Gounon P, Vincent S, Gillot I, Cuzin F (2006) RNA-mediated non-Mendelian inheritance of an epigenetic change in the mouse. Nature 441:469–474PubMedCrossRefGoogle Scholar
  41. Sutherland HG, Kearns M, Morgan HD, Headley AP, Morris C, Martin DI, Whitelaw E (2000) Reactivation of heritably silenced gene expression in mice. Mamm Genome 11:347–355PubMedCrossRefGoogle Scholar
  42. Tada M, Tada T, Lefebvre L, Barton SC, Surani MA (1997) Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells. EMBO J 16:6510–6520PubMedCrossRefGoogle Scholar
  43. Takada S, Paulsen M, Tevendale M, Tsai CE, Kelsey G, Cattanach BM, Ferguson-Smith AC (2002) Epigenetic analysis of the Dlk1-Gtl2 imprinted domain on mouse chromosome 12: implications for imprinting control from comparison with Igf2-H19. Hum Mol Genet 11:77–86PubMedCrossRefGoogle Scholar
  44. Ueda Y, Okano M, Williams C, Chen T, Georgopoulos K, Li E (2006) Roles for Dnmt3b in mammalian development: a mouse model for the ICF syndrome. Development 133:1183–1192PubMedCrossRefGoogle Scholar
  45. Velasco G, Hubé F, Rollin J, Neuillet D, Philippe C, Bouzinba-Segard H, Galvani A, Viegas-Péquignot E, Francastel C (2010) Dnmt3b recruitment through E2F6 transcriptional repressor mediates germ-line gene silencing in murine somatic tissues. Proc Natl Acad Sci USA 107(20):9281–9286PubMedCrossRefGoogle Scholar
  46. Watanabe T, Tomizawa S, Mitsuya K, Totoki Y, Yamamoto Y, Kuramochi-Miyagawa S, Iida N, Hoki Y, Murphy PJ, Toyoda A, Gotoh K, Hiura H, Arima T, Fujiyama A, Sado T, Shibata T, Nakano T, Lin H, Ichiyanagi K, Soloway PD, Sasaki H (2011) Role for piRNAs and noncoding RNA in de novo DNA methylation of the imprinted mouse Rasgrf1 locus. Science 332:848–852PubMedCrossRefGoogle Scholar
  47. Whitelaw NC, Chong S, Morgan DK, Nestor C, Bruxner TJ, Ashe A, Lambley E, Meehan R, Whitelaw E (2010) Reduced levels of two modifiers of epigenetic gene silencing, Dnmt3a and Trim28, cause increased phenotypic noise. Genome Biol 11:R111PubMedCrossRefGoogle Scholar
  48. Xu GL, Bestor TH, Bourc’his D, Hsieh CL, Tommerup N, Bugge M, Hulten M, Qu X, Russo JJ, Viegas-Péquignot E (1999) Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature 402(6758):187–191PubMedCrossRefGoogle Scholar
  49. Youngson N (2012) Challenges and future prospects for non-genetic inheritance. Nongenet Inherit 1:1–8CrossRefGoogle Scholar
  50. Youngson NA, Whitelaw E (2008) Transgenerational epigenetic effects. Annu Rev Genomics Hum Genet 9:233–257PubMedCrossRefGoogle Scholar
  51. Youngson NA, Vickaryous N, van der Horst A, Epp T, Harten S, Fleming JS, Khanna KK, de Kretser DM, Whitelaw E (2011) A missense mutation in the transcription factor Foxo3a causes teratomas and oocyte abnormalities in mice. Mamm Genome 22:235–248PubMedCrossRefGoogle Scholar
  52. Zhang Y, Jurkowska R, Soeroes S, Rajavelu A, Dhayalan A, Bock I, Rathert P, Brandt O, Reinhardt R, Fischle W, Jeltsch A (2010) Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail. Nucleic Acids Res 38:4246–4253PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Neil A. Youngson
    • 1
    • 2
  • Trevor Epp
    • 1
    • 3
  • Amity R. Roberts
    • 1
    • 4
  • Lucia Daxinger
    • 1
  • Alyson Ashe
    • 1
  • Edward Huang
    • 1
  • Krystal L. Lester
    • 5
  • Sarah K. Harten
    • 1
  • Graham F. Kay
    • 1
  • Timothy Cox
    • 6
  • Jacqueline M. Matthews
    • 5
  • Suyinn Chong
    • 1
    • 7
  • Emma Whitelaw
    • 1
    • 8
    Email author
  1. 1.Queensland Institute of Medical ResearchHerston, BrisbaneAustralia
  2. 2.Department of PharmacologySchool of Medical Sciences, University of New South WalesSydneyAustralia
  3. 3.Institute of Molecular Genetics of the ASCRPrague 4Czech Republic
  4. 4.School of Biomolecular and Physical SciencesGriffith UniversityNathanAustralia
  5. 5.School of Molecular BioscienceUniversity of SydneySydneyAustralia
  6. 6.Division of Craniofacial Medicine, Department of PediatricsUniversity of WashingtonSeattleUSA
  7. 7.Mater ResearchWoolloongabbaAustralia
  8. 8.School of Molecular Sciences, Department of GeneticsLa Trobe Institute for Molecular ScienceMelbourneAustralia

Personalised recommendations